Masood N. Khan

A method for quantitation of protein in the presence of percoll

Abstract A simple assay for protein in the presence of Percoll which requires neither additional instrumentation nor manipulation of the sample is proposed. The method is based on monitoring the absorbance at 750 and 420 nm of color developed by protein plus Percoll with Folin-Ciocalteu reagent. Protein can be determined in a sample containing as much as 30% Percoll. The method does not require the knowledge of Percoll concentration (below 30%) in the sample. A large number of samples from a Percoll density gradient can be assayed very conveniently and with great precision.

In vivo uptake of insulin into hepatic Golgi fractions: application of the diaminobenzidine-shift protocol.

The hypothesis that insulin is internalized into the hepatic Golgi apparatus was tested by the diaminobenzidine-shift protocol of Courtoy et al. (1984, J. Cell Biol. 98, 870). Highly purified Golgi fractions were isolated after the coinjection of [125I]insulin and the synthetic ligand, galactose-bovine serum albumin-horseradish peroxidase. Golgi fractions were subsequently reacted in the presence or absence of diaminobenzidine, then subjected to Percoll gradient centrifugation. For incubations carried out in the absence of diaminobenzidine, [125I]insulin-containing components were found at a low density (peak density congruent to 1.042) identical to that of the Golgi marker enzyme galactosyltransferase. However after incubations carried out in the presence of diaminobenzidine, the majority of [125I]insulin-containing components was shifted to a higher density of greater than 1.06 while that of galactosyltransferase remained unchanged (peak congruent to 1.042). These observations indicate that the majority of internalized insulin is not located in galactosyltransferase-containing Golgi components.

Insulin and Insulin Receptor Uptake into Rat Liver: Chloroquine Action on Receptor Recycling

In the present study, the effect of chloroquine on both insulin and receptor distribution was examined in vivo. Insulin injection (25 nmol/100 g body wt) caused a marked accumulation of both insulin and its receptor in purified hepatic Golgi fractions by 15 min postinjection. Percoll fractionation of parent Golgi fractions resolved two endocytic components of low (ρ = 1.040–1.050) and high (ρ = 1.053–1.064) density in which the relative distribution of insulin binding sites was unaltered by chloroquine. Chloroquine significantly accumulated in the high-density region of the Percoll gradient consistent with this being a low pH compartment. 125I-insulin accumulated first in the low-density (1 min) and subsequently in the high-density region (5–10 min) of Percollsubfractionated Golgi fractions. Chloroquine treatment caused marked accumulation of 125I-insulin in the highdensity compartment with substantial retention of radiolabel therein at 20 min postinjection. 125I-insulin extracted from the Percoll fractions was comparably intact in control and chloroquine-treated rats. These data suggest that the chloroquine-accumulating, high-density compartment of hepatic Golgi fractions is the site of dissociation of internalized insulin-receptor complexes before degradation of the ligand and receptor recycling.

Prolactin uptake into liver endocytic components. Reduced sensitivity to chloroquine

Subfractionation of hepatic Golgi fractions on Percoll gradients revealed two populations of lactogen-receptor enriched components with one pool of rho = 1.040-1.050 and a high-density component of rho = 1.053-1.064. 125I-labelled prolactin uptake into Percoll gradient subfractions demonstrated rapid accumulation into low-density elements and slower accumulation in high-density structures. Electron microscope radioautography demonstrated that silver grains were largely associated with lipoprotein-filled structures. Radiolabel was highly concentrated in these components especially the very pure higher-density component where enrichment over the homogenate was 184-fold at the peak time after injection (10 min). Administered chloroquine accumulated in the higher-density component but not in low-density elements, suggesting that the former are at an acid intraluminal pH. In contrast to the marked effect of chloroquine on insulin uptake into endocytic structures found in Golgi fractions (Posner, B.I., Patel, B.A., Khan, M.N. and Bergeron, J.J.M. (1982) J. Biol. Chem. 257, 5789-5799), little effect of this pH disrupting agent was observed on prolactin uptake. The difference between the effect of chloroquine on 125I-labelled prolactin and 125I-labelled insulin uptake may reflect the greater stability of prolactin-receptor complexes in a low-pH environment.

Potential Role of Endosomes in Transmembrane Signaling

After ligands bind to their surface receptors on target cells, the ligand-receptor complexes undergo aggregation and internalization. This process of receptormediated endocytosis leads to the concentration of ligand-receptor complexes in a heterogeneous population of tubulovesicular structures that has been distinguished from plasma membrane, lysosomes, and Golgi elements. These structures, now called endosomes, are not clearly characterized by a specific enzyme or protein marker but rather by their unique property to concentrate internalized ligand-receptor complexes. It is widely appreciated that, within endosomes, ligand-receptor complexes are directed toward dissociation and receptor recycling or toward transcytosis, with ligand undergoing degradation in the former situation or transcellular passage in the latter. It has been suggested that, in addition to this sorting function, endosomes play a role in signal transduction.

Peptide hormone receptors in intracellular structures from rat liver.

Publisher Summary This chapter presents methods that are used in laboratories for the preparation and analysis of peptide hormone receptor-rich structures derived from within the rat hepatocyte. A new entity—the intermediate or unique vesicle—has been observed to contribute to the heterogeneity of Golgi fractions. Sherman or Sprague–Dawley rats 150 to 250 g each have been used in these studies. The animals are fed ad libitum to the day before use and are fasted overnight before the experiment. They are killed by decapitation and exsanguinated before the livers are removed. All sucrose solutions are prepared with reagent grade material. The chapter explains that in earlier studies the rats received ethanol (0.6 g/100 g body wt) in a 50% (w/v) solution by stomach tube 90 minutes prior to sacrifice. In more recent studies, the use of ethanol has been discontinued.

The Endosomal Apparatus and Transmembrane Signalling

The binding of insulin, prolactin, and epidermal growth factor to their respective receptors on liver parenchymal cells rapidly leads to their internalization along with their associated receptors. As reviewed elsewhere, 1–5 high concentrations of these ligand-receptor complexes accumulate initially in a heterogeneous group of compartments collectively termed the endosomal apparatus. Conceptually, the endosomal apparatus bridges the plasmalemma with secondary lysosomes and involves at least indirectly the Golgi apparatus (Fig. 1 and 14–17). In liver, components of the endosomal apparatus have been visualized and identified by electron microscope radioautography, 18 EM immunocytochemistry, 19–23 and subcellular fractionation 7–11,24. These techniques have demonstrated that the endosomal components are not lysosomes, 10,12,18,25 that a sequential transport of ligands and receptors among endosomal components can be observed, 8,9,26,29 and that morphologically different microdomains can be observed in continuous structures segregating ligands (vesicular) from receptors (tubular)19–21.

Internalization of hormone receptor complexes: route and significance.

Receptor-mediated endocytosis is a general process by which a wide variety of ligands are taken up with high specificity and affinity into cells. In this review we shall primarily discuss data in regard to the internalization of peptide hormones, especially insulin, but it is noteworthy that there are similarities as well as interesting differences by which membrane-bound lectins, immune complexes, viruses, toxins, etc. are handled by cells (Bergeron et al., 1985).

Internalization of Insulin and Its Receptor: Role in Signaling

Tissue resistance to the action of insulin plays a role in the pathogenesis of Diabetes Mellitus, especially the Type II variety.1 This has given clinical relevance to investigation of the mechanism of insulin action on target tissues. One approach to studying insulin action is to examine the nature of its interaction with target cells. In this article we shall briefly outline the fate of insulin following interaction with its receptor, and the role that subsequent cellular handling of insulin-receptor complexes might play in hormone signal transmission.

Insulin Internalization as Studied by Subcellular Fractionation and Electron-Microscopic Radioautography

The techniques of subcellular fractionation and e.m. radioautography have defined novel intracellular components which participate in receptor-mediated endocytosis [1, 2]. These components have been interpreted as vesicles accumulating within the trans* Golgi apparatus (G.a.). The vesicles are highly enriched in receptors for insulin and prolactin; during receptor-mediated insulin internalization the vesicles concentrate reasonably intact insulin prior to donation of hormone to a catabolic compartment (probably the secondary lysosome).